Targeted Next Generation Sequencing Identified Novel Mutations In

www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 21), pp: 35176-35183

Research Paper Targeted next generation sequencing identified novel mutations in RPGRIP1 associated with both retinitis pigmentosa and Leber's congenital amaurosis in unrelated Chinese patients

Hui Huang1,*, Ying Wang2,3,*, Huishuang Chen1,*, Yanhua Chen1,4, Jing Wu1, Pei- Wen Chiang5, Ning Fan2, Yan Su1, Jianlian Deng1, Dongna Chen1, Yang Li1, Xinxin Zhang1,6, Mengxin Zhang7, Shengran Liang1, Santasree Banerjee1, Ming Qi1,8,9 and Xuyang Liu2,10 1BGI-Shenzhen, Shenzhen, China 2Shenzhen Key Laboratory of Ophthalmology, Shenzhen Eye Hospital, Jinan University, Shenzhen, China 3Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, China 4School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China 5Casey Eye Institute Molecular Diagnostic Laboratory, Portland, Oregon, USA 6BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China 7Department of Applied Biology with Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong 8School of Basic Medical Sciences, Zhejiang University, Hangzhou, China 9Functional Genomics Center, Department of Pathology & Laboratory Medicine, University of Rochester Medical Center, West Henrietta, New York, USA 10School of Ophthalmology & Optometry, Shenzhen University, Shenzhen, China *These authors have contributed equally to this work Correspondence to: Xuyang Liu, email: [email protected] Ming Qi, email: [email protected] Keywords: targeted next generation sequencing, gene panel, novel mutation, retinitis pigmentosa, Leber's congenital amaurosis Received: October 26, 2016 Accepted: March 15, 2017 Published: April 12, 2017 Copyright: Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT

As the most common inherited retinal degenerations, retinitis pigmentosa (RP) is clinically and genetically heterogeneous. Some of the RP genes are also associated with other retinal diseases, such as LCA (Leber's congenital amaurosis) and CORD (cone-rod dystrophy). Here, in our molecular diagnosis of 99 Chinese RP patients using targeted gene capture sequencing, three probands were found to carry mutations of RPGRIP1, which was known to be associated with pathogenesis of LCA and CORD. By further clinical analysis, two probands were confirmed to be RP patients and one was confirmed to be LCA patient. These novel mutations were co-segregated with the disease phenotype in their families. Our result not only expands the mutational spectrum of the RPGRIP1 gene but also gives supports to clinical diagnosis and molecular treatment of RP patients.

INTRODUCTION associated with the rod photoreceptors, while the cone cells are compromised as the disease progresses [3]. RP Retinitis pigmentosa (RP) is the most common individuals exhibit degeneration of photoreceptors or the inherited retinal dystrophy, affecting approximately 1 retinal pigment epithelium (RPE) of the retina in the initial in 5,000 individuals worldwide [1, 2]. RP is primarily stages of the disease, followed by a progressive reduction www.impactjournals.com/oncotarget 35176 Oncotarget in the visual field and visual loss [3]. Other ocular findings fluorescence existed in the corresponding pigmentary area include atrophic changes in the photoreceptors and RPE in the inferior periphery retina of the right eye. The normal followed by the appearing of melanin-containing structures structure of macular arch was absent (Figure 1d). Full-field in the retinal vascular layer [3]. The fundus changes include electroretinography (fERGs) disclosed no detectable rod a pale optic nerve, attenuated retinal vessels, and bone responses to single flashes of blue light and cone responses spicule-like pigmentation in the mid-peripheral retina [3]. to the 30 Hz flicker that were reduced 99% and delayed RP is phenotypically and genetically heterogeneous (Figure 1e & Supplementary Figure 1). All the status of with almost 60 different genes identified till now, and could severe retinal structure abnormity and function loss support be divided into the early onset RP which exhibits symptoms the clinical diagnosis of RP. around 2 years old and the late onset RP which may not Patient P024, as diagnosed with RP is a 22 years old develop symptoms until mid-life [2]. The clinical distinction female with progressive vision weakness for 20+ years. between the subtypes of RP, however, is not always clear She is the only patient in a non-consanguineous family. and there are largely overlapped in phenotype between RP, Nystagmus was noticed in her first 8 months, and then LCA (Leber’s congenital amaurosis), COD or CORD (cone gradual loss of visual acuity and night blindness appeared. or cone-rod dystrophy) and other retinal diseases [3]. Also, In the last 6 years, her visual field showed losses of there are other non-RP diseases caused by mutations in the peripheral vision, and eventually became tunnel vision same genes involved with RP. With the development of in the age of 21. Her BCVA was 12/400 with both eyes. sequencing technology, more disease-causing genes have The dilated fundus examination showed attenuated retina been identified, 60 for RP, 20 for LCA and 29 for CORD vascular with absent pigmentary changes (Figure 1f). The respectively. Among them, 9 genes (CRB1, CRX, LRAT, ERG performed extinguished waveform, which indicated PRPH2, RDH12, RPE65, SPATA7, TULP1, IMPDH1) that the rod cell and cone cell were severely damaged were previously reported to be associated with both RP (Figure 1g). and LCA (RetNet). However, large number of RP patients Patient P030 is female, 36 years of age, who was is waiting for uncovering their specific genetic underlying diagnosed as LCA. She was born with severe visual causes. In our recent molecular screening of 99 RP patients, impairment and nystagmus without any other symptoms. two RP and one LCA patients were found to carry mutated Her BCVA was hand motion in the age of 36. Dilated fundus RPGRIP1, which was previously reported to be implicated examination revealed narrowed arterioles and tortuous veins. in LCA and CORD [4, 5]. Obviously bone corpuscle types pigmentary were seen in the periphery retina (Figure 1h). Scotopic ERG (rod response) RESULTS after 30min dark adaptation showed the extinguishment of the a-wave and b-wave in both eyes. Only three waves of Clinical findings oscillatory potential ERG were recorded, their amplitudes were severe declined. Cone system was affected with slightly Patient P065 is a 30 years old male with early-onset delayed b-wave implicit time. He exhibited the absence of RP. He exhibited early-onset bilateral blurred vision in normal ERG response to a 30Hz flickering light (Figure 1i). childhood (visual acuity: 20/25), nystagmus and night Fundus fluorescence angiography (FFA) showed the edges blindness at about 23 years old based on his recall. Then of optic disks were vague. The illumination of periphery he gradually presented with decreasing vision and loss of scattered transmitted fluorescences was gradually increasing. peripheral visual fields. At 30 years of age, his best corrected Patchy blocked fluorescence existed in the periphery visual acuity (BCVA) was 20/133 with -2.25 diopter in the area. Vessels obstruction induced massive periphery non- right eye and 20/200 with -2.00 diopter in the left. Fundus perfusion area (Figure 1j). examination revealed waxy disc, obviously attenuated retina vascular. No significant pigmentary changes of salt Molecular diagnosis and validation and pepper or bone corpuscle type were noted in the left eye. A few patchy pigmentary could be seen inferior in We performed next generation sequencing on these the periphery retina (Figure 1a). Center 30° visual field 3 patients using the panel described in Methods section. examination (Topcon SBP-300) showed tubular vision 466, 362, and 351 fold of depth, 94.26%, 94.31%, and with little center visual island left (Figure 1b). Time- 94.04% of coverage were obtained from the 3 patient dominant optical coherence tomography (TD-OCT) samples (Supplementary Table 1), respectively, indicating showed significant thinner outer retina, especially in the left that sufficient depth and coverage were obtained for eye (Figure 1c). Fundus fluorescence angiography (FFA) variants calling. We identified 8, 15, 8 rare variants (rare: showed a significant delay in the arm-retina circulation frequency < 0.01 in 1000genome, dbSNP, HGMD and time (ART) from 10~15sec to 17sec of the left eye, which our 200 control local database) that would lead to protein exhibited considerable attenuation of the retinal arterioles. coding change (including potential splice site in 10bp of Absent peripheral background fluorescence indicated exon/intron borders) in the 283 eye disease related genes that choriocapillaris were atrophied. Patchy blocked (Supplementary Table 2). www.impactjournals.com/oncotarget 35177 Oncotarget We found these 3 patients carrying mutations in the HapMap and 1K genome and was co-segregated not in RP related genes but in gene RPGRIP1 which with the disease phenotype in the family; the normal was previously reported to cause Leber’s congenital family members carried only one heterozygous mutant amaurosis [4] and Cone-rod dystrophy [5]. Among them, allele (Figure 2a & Figure 2b). one patient carried compound heterozygous mutations This RPGRIP1 mutation disrupts the splice acceptor and 2 patients carried homozygous mutations (Table 1). site of exon 12. With this type of splicing defect, it would Sanger sequencing and quantitative PCR was undertaken be desirable to determine the consequences at the mRNA to validate all these 4 RPGRIP1 mutations in the proband level by reverse transcription-PCR (RT-PCR) of RPGRIP1 and their family members (Figure 2). cDNA. Partial genomic DNA constructs consisting of Patient P065 was detected to carry one novel exons 12 and partial intron 11 and intron 12 of RPGRIP1, homozygous splicing site mutation c.1468-2A>G in gene with or without the splice site mutation, were expressed in RPGRIP1, his parents were consanguineous with normal COS-7 cells. RT-PCR and direct sequencing of RPGRIP1 phenotype. This homozygous mutation was not recorded cDNA from cells transfected with wild-type construct

Figure 1: Clinical characteristics of 3 patients Patient P065. (a) Fundus photographs showed disc pallor with wax yellow. Obviously attenuated narrow arterioles and tortuous veins lay on the orange background of the retina. (b) vision field examination (Topcon SBP-300) show tubular vision with litter center visual island. (c) OCT showed significant thinner of outer layer of retina, especially in the left eye. (d) Fudus fluorescence angiography (FFA) showed a significant delay in the arm-retina circulation time (ART) from 10~15sec to 17sec of the left eye. There were uniform scattered dotted transmitted fluorescences on the both side in the late phase. Absent peripheral background fluorescence indicted choriocapillaris were atrophy. Patchy blocked fluorescence existed in the corresponding pigmentary area in the inferior periphery retina of the right eye. The normal structure of macular arch was absent. (e) Scotopic ERG (rod response) and dark adapted bright flash ERG showed a-, b-waves extinguished in both eyes. The patient exhibited extinguished in the ERG response. Patient P024 (f) Fundoscopy revealed narrowing of retinal vessels. Scattered patchy dislocatedpigmentary changes could be seen in the periphery retina of both eyes. (g) ERG revealed extinguished waveforms. Patient P030 (h) Dilated fudus examination revealed arterioles and tortuous veins narrowed. Patient’s optic disk was sanguineness. Obviously bone corpuscle types pigmentary were seen in the periphery retina. (i) Scotopic ERG (rod response) after 30min dark adaptation showed the extinguishment of waveform in both eyes. B-wave of photopic ERG (cone response) after 10min light adaptation showed implicit time delay to 49ms which was amplified to severe decline in both eyes. Patient exhibited the absence of nomal ERG response to a 30Hz flickering light. (j) FFA showed Retinal arterioles were narrowed. The edges of optic disks were vague. The illumination of periphery scattered transmitted fluorescences was increasing gradually. Patchy blocked fluorescence existed in the periphery area. Vessels obstruction induced massive periphery non-perfusion area. www.impactjournals.com/oncotarget 35178 Oncotarget Table 1: RPGRIP1 mutations identified in three families Patient ID Disease Type Exon Mutation Protein change 65 ARRP Homozygous Intron11 c.1468-2A>G Exon12 del Compound c.154C>T/ p.Arg52*/ 24 ARRP Exon 2 / Exon 14 heterozygous c.2020C>T p.Pro674Ser 30 LCA Homozygous Exon1-22 ex1-22del

RP: Retinitis Pigmentosa; LCA: Leber congenital amaurosis.

showed normal splicing of exon 12. In contrast, direct DISCUSSION sequencing of mutant RT-PCR products revealed complete loss of exon 12 resulting removal of 48 amino acids in In this study, we found 2 RP patients and 1 LCA the RPGRIP1 polypeptide due to abolition of the acceptor patient carrying mutations in RPGRIP1 using our splice site (data not shown). Prediction of the abolition panel NGS methods. One RP patient carried a novel effect of this splice site was made using a splice predictor homozygous mutation of c.1468-2A>G in intron 11 program, Human Splicing Finder (http://www.umd.be/ of RPGRIP1, resulting in the formation of alternative HSF). This prediction gave a potential breakage of splice transcripts and leading to the complete loss of exon 12 site (ttccctctacggCC), compared with a probability of (data not shown). Defects in either RPGRIP1 or its RPGR 92.13 for the wild type (ttccctctacagCC) splice site. All interacting protein probably alter a functional complex in the results suggest that aberrant splicing caused by this the connecting cilia of rods and cones. Another RP patient mutation is quite efficient as it completely abolishes carried compound heterozygote for c.154C>T (p. Arg52*) normal splicing in the mutant allele. and c.2020C>T (p. Pro674Ser). The LCA patient carried Patient P024 carried one nonsense mutation a large homozygous deletion that extends from exon 1 to c.154C>T (p. Arg52*) and one novel missense mutation intron 22 in RPGRIP1. All these mutations were confirmed c.2020C>T (p. Pro674Ser) in gene RPGRIP1. Mutation to be co-segregated with disease phenotype in respective p. Arg52* was inherited from the father and mutation families by Sanger sequencing or quantitative PCR. c.2020C>T (p. Pro674Ser) came from the mother This study provides evidence supporting that (Figure 2c & Figure 2d). The nonsense mutation p. RPGRIP1 is a new RP-causing gene by finding mutations Arg52* was previously reported in a retinitis punctata in 2 unrelated cases. We also found that the LCA patient albescens (RPA) patient [6]. It creates premature stop carrying mutation in RPGRIP1 presented with similar codons and is likely to be a null allele. The mutation p. phenotype. In clinical signs and symptoms, all these 3 Pro674Ser is novel and not recorded in the HapMap and patients presented with early-onset night blindness and 1K genome database or our local database, it changed a low visual acuity. According to the clinical examination proline (non-polar, uncharged amino acid) into a serine and genetic test of these 3 patients, it suggested a (polar) in the protein. In addition, it was predicted to correlation between disease severity and the nature of be “probably damaging” with a score of 1.000 using the mutations. Patient P030 carrying a large exon1-22 PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/). deletion mutation in RPGRIP1 showed the most severe The proline at position 674 is present as a highly phenotype of three patients, she was blind with light conserved amino acid among mammals (Figure 2e). It is perception at birth and her current BCVA was hand highly likely that these mutations are the primary cause motion, and she was diagnosed with LCA. Patient P024 of the retinal disease in this patient. and P065, who were diagnosed with RP, showed slighter Patient P030 was found carrying no SNV/InDel symptoms than Patient P030. Patient P024 carrying a mutation, so we tried to search a CNV (copy number nonsense (p. Arg52*) and a missense mutation (p. variantion) from exonic depth and coverage in retinal genes, Pro674Ser) showed a worse BCVA comparing to Patient and a large homozygous deletion of exon 1-22 in RPGRIP1 P065 who carried a homozygous splice site mutation was found (Supplementary Figure 2), which was inherited (c.1468-2A>G). It is possible that the large proportion from her healthy parents separately (Figure 2f). The of coding region deletion and nonsense mutation in the subsequent quantity PCR confirmed this mutation (Figure initiating terminal coding region leads to loss of the 2g). This large deletion may lead to absence of RPGRIP1 whole protein, while the missense mutation and the protein which interacts with RPGR in the connecting of splice site mutation (although it resulted in the complete cilia of rods and cones [7]. This homozygous mutation was loss of exon 12) retains most of protein residue. not identified in the HapMap and 1000 genome and was co- The RPGRIP1 gene contains 24 exons and segregated with the disease phenotype. Her normal brother encompasses three main domains that are RPGR didn’t carry the mutation (Figure 2g). interacting domain (RID), coiled-coil (C2) domains and www.impactjournals.com/oncotarget 35179 Oncotarget a domain of unknown function. The RPGRIP1 protein is two disorders are characterized by LCA/RP and other localized in the connecting cilia of human cone and rod systemic features including kidney conditions [10–11]. photoreceptors that connect inner and outer segments, These studies suggest that RPGRIP1 is important in visual where it binds RPGR to the cilium. Mice lacking pathway and has an essential function in eye development, RPGRIP elaborate grossly oversized outer segment disks and when mutated, it may cause RP disease. resembling a cytochalasin D-induced defect and have Interestingly, RP is both clinically and genetically a more severe disease than mice lacking RPGR. Mice heterogeneous. The phenotype of such patients is overlap lacking both proteins are phenotypically indistinguishable between retinal diseases, as RP and LCA is more overlap from mice lacking RPGRIP alone [8]. RPGRIP1 has an in the initial stage, while RP and CORD is in the end stage interaction not only with RPGR, but also with NPHP4 [3]. There are nine genes (CRB1, CRX, LRAT, PRPH2, and SDCCAG8, Rpgrip1nmf247 mice without RPGRIP1 RDH12, RPE65, SPATA7, TULP1, IMPDH1) overlap expression lack NPHP4 and RPGR in photoreceptor cilia, between RP and LCA, eight genes (CRX, PROM1, whereas the SDCCAG8 and acetylated-a-tubulin ciliary PRPH2, SEMA4A, ABCA4, C8orf37, CERKL, RPGR) localizations are strongly decreased, even though the between RP and COD/CORD (RetNet: https://sph.uth. NPHP4 and SDCCAG8 expression levels are normal and edu/retnet/sum-dis.htm). CRX and PRPH2 are the only those of acetylated-a-tubulin and c-tubulin are upregulated two genes associated with RP, LCA and CORD. Carlo [9]. Mutations in NPHP4 and SDCCAG8 would cause Rivolta et al. analyzed 18 mutations and Huang L et al. Senior-Loken syndrome and Bardet-Biedl syndrome, these also reviewed 49 mutations in CRX, but they all could not

Figure 2: Molecular genetic test. (a) The pedigree of Patient P065; (b) Sanger sequence chromatograms for the pedigree of Patient P065; (c) The pedigree of patient P024; (d) Sanger sequence chromatograms for the pedigree of Patient P024; (e) Evaluation of the novel RPGRIP1 missense mutation in Patient P024. Multiple alignments using MEGA6 software (Tamura K et al. 2013) and amino acid conservation of the novel missense sequence variants were performed. The alignment results showed that proline at codon 2020 were fully conserved through all species. (f) The pedigree of patient P030; (g) Quantity PCR for the large deletion in pedigree of Patient P030.

Figure 3: Reported variants in RPGRIP1 identified in individuals with LCA, CORD and RP. www.impactjournals.com/oncotarget 35180 Oncotarget uncover any correlation between phenotype and genotype representatives have given permission for the mutation [12–13]. In this study, we also summarized the mutations detection. Informed consents were also obtained from in RPGRIP1 which were reported to be related to LCA patients. This study was approved by the institutional or CORD as many as we can (Figure 3). We can see that review board on bioethics and biosafety of BGI, and the approximately 90% of the reported mutations causing LCA IRB approval number is BGI-IRB 14002. in RPGRIP1 were frameshift or nonsense mutations that will lead to create a prematurely truncate protein, which DNA preparation are expected to abolish the function of RPGR interacting domain, even the RPGRIP1 function. RPGRIP1 was Venous blood samples were obtained from the found to cause LCA in 2001 [4], then in 2002, Cremers probands and their family members. Genomic DNA FP et al. deduced that mutations allow residual RPGRIP1 was extracted from 200μl peripheral venous blood by activity may result in less severe phenotypes, such as standard procedures using QIAmp DNA Blood Mini kit RP or CORD, than LCA [14]. Subsequently in 2003, it (QIAGEN, Hilden, Germany) according to recommended was proved that homozygous missense mutations in instructions. DNA integrity was evaluated by 2% agarose RPGRIP1 would cause CORD [5]. In 2005, J C Booij et gel electrophoresis. All DNA samples were stored at -20°C al. found two RP patients carrying respective frameshift after the analysis with NanoDrop 2000 (Thermo Fisher and missense mutations in RPGRIP1, but it’s a pity that Scientific, Waltham, USA). they identified only one heterozygous mutation in every patient [15]. Yet, in this study, we found two RP patients Panel sequencing library preparation and caused by homozygous splice site mutation and compound sequencing heterozygous (missense and nonsense) mutations in RPGRIP1. Taken together, it showed strong evidence for 283 genes, which include 164 known retinal disease- the conclusion that RPGRIP1 is a causative gene for RP. causing genes, were collected by systematic database In the past, people always found new disease- (GeneReviews(®), OMIM, and RetNet), literature causing genes by linkage study and subsequent whole searches, and expert reviews (Supplementary File 1). exome sequencing (WES). In this study, we found a new Customized oligonucleotide probes were designed to RP disease-causing gene by panel sequencing. Although capture exons and adjacent 30bp sequences of above genes all the captured genes were known hereditary eye disease- using NimbleGen (Roche) online oligonucleotide probe causing genes, many different retinal disease genes are design system. Target sequencing libraries were prepared in the same pathway (KEGG), it provides the potential as below: 1μg genomic DNA was sonicated to 200~300 possibility that one gene in this pathway causes several bp fragments, followed with end-repair, A-tailing, retinal diseases. So we can find a new gene for some Illumina adaptors ligation, 4 cycles of pre-capture PCR retinal diseases using a panel that capture genes in the amplification and sample indexing. Then the indexed PCR pathway. product of 20-30 samples were pooled, targeted capture In conclusion, we reported that the mutations in was performed by hybridizing with capture probes, RPGRIP1 causes early-onset RP based on the data from and followed by 15 cycles of PCR amplification. DNA panel NGS, co-segregation, OCT, histopathological and sequencing was done on Illumina HiSeq2500 sequencer electrophysiological analysis. Thus our findings suggest to generate paired-end reads including 90 bps at each end that RPGRIP1 gene can also be classified into the RP and 8 bps of the index tag. And bases were called by the causative genes, that expands the clinical spectrum of Illumina build-in Pipeline. RPGRIP1 related disease. Since the two RP patients in this study were found in a cohort of 99 unselective Bioinformatics analysis and data filtering Chinese patients, we can expect that RPGRIP1 would cause about 2% RP patients in Chinese population. In Reads filtering was surveillance by indexed primers. addition, RPGRIP1 has been reported with effective The reference genome was downloaded from the NCBI, treatment in animal models and showing molecular version GRCh37 (hg19). Sequence was aligned to the treatment potential in a LCA patient [16, 17]. Hope our reference using Burrows Wheeler Aligner (BWA) Multi- research could improve the diagnosis and treatment of RP Vision software package [18]. SNVs were called using and LCA. SOAPsnp [19], while small InDels (<20bp) were called using the Samtools (Tools for alignments in the SAM format) Version: 0.1.18, http://samtools.sourceforge.net/. MATERIALS AND METHODS The threshold for filtering SNPs included the followings: 1) the consensus quality score had to be >= 20 (The Patients quality score is a Phred score, generated by the program This study was performed in agreement with the SOAPsnp, quality score 20 represents 99% accuracy of declaration of Helsinki. All the patients and their legal a base call); 2) the number of uniquely mapped reads supporting a SNP had to be >= 4; 3) the estimated copy www.impactjournals.com/oncotarget 35181 Oncotarget number is no more than 2; 4) the distance between two ACKNOWLEDGMENTS SNPs should be larger than 5. This work was supported by grants from the National Sanger sequencing Natural Science Foundation of China (NSFC81500718 and 81371009), the Science and Technology Innovation Mutations identified in Target sequencing were Committee of Shenzhen (JCYJ20160428144701106) and validated by Sanger sequencing. Primers flanking the Shenzhen Municipal Science and Technology Plan Project candidate loci were designed based on reference genomic (No.201402089, 201507044). We thank all the patients sequences of Human Genome from gene bank in NCBI and their families for their participation. (GRCh37, hg19) and synthesized by Invitrogen (Shanghai, China). PCR amplification was carried out in ABI 9700 CONFLICTS OF INTEREST Thermal Cycler. Subsequently, all PCR products were sequenced on ABI PRISM 3730 automated sequencer The authors declare no conflicts of interest. (Applied Biosystems).

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